Overcoming FFPE Challenges with a Scalable, Automated Workflow
At ASHG 2022, Jennifer Schroeder, Automation Development Specialist at Covaris, presented an Adaptive Focused Acoustics ® (AFA®) Technology-enabled workflow that can be partially or fully automated to help labs overcome major challenges in FFPE tissue extraction and purification, including low sample quality, health and safety concerns from toxic chemicals, and very high demand.
Introduction
For decades, advances in precision medicine have depended on the extraction and analysis of DNA and RNA from formalin-fixed paraffin embedded (FFPE) tissue samples. Because FFPE samples can be stored at room temperature while preserving morphological and cellular details, they continue to be widely used by researchers and clinicians.
Moreover, disease and treatment response monitoring strategies in oncology and other fields require periodic sampling of tissue from the same patient. The resulting high demand for FFPE tissue processing has created a massive backlog of samples. To process them in a timely and cost-effective manner, better and less laborious workflows that can accommodate higher throughputs are sorely needed (Figure 1).
In this talk, a novel, high throughput FFPE extraction approach that can solve these challenges is discussed. The Covaris Adaptive Focused Acoustics (AFA) Technology-enabled workflow can be partially or fully automated and was designed to help labs overcome low sample quality, health and safety concerns from toxic chemicals, and high demand.
Conventional FFPE Tissue Processing Poses Challenges to Efficiency
Researchers and patients depend on critical insights derived from FFPE tissue analysis. However, conventional existing workflows are limited by several challenges to reproducibility, reliability, and robustness:
- Low Sample Quality: Passive deparaffinization, despite being time-consuming, does not remove wax completely, and results in some tissue that is still shielded from Proteinase K digestion. This step also requires harsh organic solvents, which can degrade RNA and prevent optimal recovery for library prep protocols like TruSight® Oncology 500.
- Safety and Workflow Complications: Harsh organic solvents like xylene and its derivatives are highly toxic and require dedicated workspaces, environmental health and safety training, and waste disposal considerations. As a result, workflows that involve these solvents are typically costly and difficult to automate.
- Sheer Quantity: An enormous number of new FFPE samples are collected daily, prompting labs to consider automation and scalability while maintaining compatibility with existing processes and platforms.
Figure 1. The FFPE tissue samples market is projected to continue growing, requiring innovative automated solutions to improve processing efficiency (Image source: https://www.transparencymarketresearch.com/ffpe-tissue-samples-market.html).
The Covaris Solution Offers Automation-Friendly Deparaffinization and Extraction
To help labs overcome issues of quality, safety, and time, Covaris developed the truXTRAC® FFPE Total Nucleic Acid Auto 96 Kit—or Auto 96 for short. Leveraging Covaris’ AFA technology via the R230 Focused-ultrasonicator, this workflow simultaneously deparaffinizes samples, emulsifies paraffin, and rehydrates tissue—all without harsh organic solvents. As a result, it steers clear of issues like formaldehyde crosslinks, mixtures of single and double stranded DNA, and low amounts of nucleic acid.
To simplify workflows further, the Auto 96 Kit and R230 Focused-ultrasonicator can be integrated with many liquid handlers. Unlike other solutions, this enables full automation of both extraction and purification steps (Figure 2).
Figure 2. Advantages of the Covaris Auto 96 workflow vs. other available methods.
The Auto 96 Workflow Delivers a Streamlined and Flexible Automation Integration
The Auto 96 workflow begins by adding rehydration buffer and deparaffinization solution to FFPE samples in truTUBES. The samples are then incubated and centrifuged before the supernatant is removed. At this point, an additional AFA-enabled tissue homogenization step scrubs the remaining paraffin out of the sample prior to Pro-K digestion and centrifugation.
Here, decrosslinking and magnetic bead purification produce fully extracted DNA on one side and RNA on the other. A number of steps in the workflow can be performed manually or automated, offering flexibility for evaluating performance (Figure 3).
Figure 3. The Covaris truXTRAC FFPE Total NA Auto 96 Kit workflow, including optional manual steps.
Covaris aimed to first automate about 70% of the Auto 96 workflow using the Dynamic Devices® Lynx®, a smaller liquid handler (Figure 4). TNA, RNA, and DNA portions were scripted separately. To reduce the significant amount of capping/decapping time needed for a 96-sample format, a LabElite® DeCapper was added on deck. The R230 Focused-ultrasonicator was accommodated on 4 deck positions.
Figure 4. A semi-automated Auto 96 workflow on the deck of the Dynamic Devices Lynx.
Fully Automated Assay Ready Workstation Built in Collaboration with Hamilton®
While 70% automation was a good start in terms of reducing hands-on time and errors while boosting reproducibility, Covaris embarked on a partnership with Hamilton to develop a fully automated workflow on the Hamilton STAR.
First, separate manual and automated extraction kits were created to enable customers to evaluate performance manually at a lower throughput before switching to automated kits with 96-well plates. To evaluate performance of the kits, customers can rent or purchase the manual accessory kits before switching to the auto kits once automation is implemented. Both kits contain similar contents, but different manual accessories are required to accommodate testing of 1-16 samples in a manual workflow.
Customers are required to provide their own 4000 rcf or higher centrifuge, 8 and 12 channel reservoirs, Q Instrument heat plates, and an Alpaqua magnetic rack.
The assay ready workstation (ARW) built in collaboration with Hamilton incorporates the entire Auto 96 Kit workflow and all Covaris accessories to power simplified, efficient FFPE tissue sample processing (Figure 5).
Figure 5. Hamilton STAR Deck Layout for Full Automation of Auto 96 Kits.
Covaris Builds Custom Automation Solutions to Meet Unique Needs
In recognition that one approach doesn’t fit all, Covaris aims to provide a broad range of agnostic modular options tailored to labs who want to leverage existing equipment and workflows.
For example, one Covaris collaborator wanted to build out a stepwise integration to maximize peripheral integration ROI to optimize the versatility of their liquid handler. They were interested in starting with automating pipetting steps on the STAR and using accessories off-deck.
Covaris created an automation plan that met the collaborator’s implementation schedule and scalability goals. The plan began by calculating the time and effort-saving value of each peripheral in the Auto 96 workflow to build a roadmap from partial to full integration to support increasing throughput needs.
The roadmap to full automation involved four phases (Figure 6):
- Integrating the liquid handler and magnetic rack while using everything else off-deck.
- Adding the Capper/Decapper on deck.
- Adding all other components minus the centrifuge.
- Achieving a custom full walkaway automation solution that includes the centrifuge and plate sealer.
Figure 6. A roadmap from partial to full automation for a Covaris collaborator.
Another collaborator aimed to add a semi-automated Auto 96 workflow to an existing Hamilton STAR integrated with a KingfisherTM Presto Purification System. Covaris worked to leverage the Presto for the purification steps and minimize modifications to the deck so they could continue using it with other workflows.
Conclusion
As the demand for FFPE tissue processing continues to grow, more labs will need to implement more efficient and less time-intensive automated workflows. The truXTRAC FFPE Total Nucleic Acid Auto 96 Kit from Covaris harnesses AFA technology to achieve more complete paraffin removal without the complications of harsh or organic solvents, while enabling partial or full automation. Covaris automation specialists work with labs to build and deploy custom automation solutions for specific equipment and workflow needs.
Are you interested in discussing the truXTRAC FFPE Total Nucleic Acid Auto 96 Kit, AFA technology, or automation solutions with Covaris? Click here to reach out to one of our specialists!